Oncogenic Foxl2 is a chromatin-remodeling pioneer transcription factor in adult-type ovarian granulosa cell tumors Veena K. Vuttaradhi a , Eleonora Khlebus a , Thomas Welte a , Namrata Khurana a , R. Tyler Hillman a,b,c a Department of Genomic Medicine; b Department of Gynecologic Oncology and Reproductive Medicine; c CPRIT Scholar in Cancer Research-The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
Background • Adult-type granulosa cell tumors (aGCTs) are rare sex-cord stromal tumors that account for 5% of total ovarian cancers 1 . • A unique missense point mutation in the Forkhead domain-containing FOXL2 (Foxl2 p.C134W) transcription factor is pathognomonic for aGCTs 2,3 , but the oncogenic mechanism of this mutation is not known. • Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins 4 (Fig.1).
Results • Using the allele-specific CRISPR-Cas9 method, two specific, novel KGN- FOXL2 -/- and KGN-FOXL2 -/C134W isogenic cell lines were isolated (Fig. 3 A & B). • Intense negative selection against the inactivation of FOXL2c.C402G was observed only in 1% of KGN-FOXL2 edited lines. • Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high- confidence peaks. De novo motif discovery performed on Foxl2-C134W ChIP-seq peaks identified the canonical Foxl2 binding motif (P = 1.4 x 10 -7 ) in 44.9% of peaks but also identified a novel variant motif (P = 6.7 x 10 -15 ) in 68.5% of peaks (Fig 4 A). • Median chromatin accessibility at Foxl2- C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 x 10-16 for both comparisons) (Fig 4 B). • No difference was observed between SKO and parental cell lines (P = 0.19). • Decreased chromatin accessibility at Foxl2- C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Conclusions • Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements. • The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re- directing this pioneering function to sites containing a novel variant Foxl2 binding motif.
Fig. 3A Allele-specific Sanger sequencing data of FOXL2- edited KGN- lines; B Foxl2 expression across FOXL2-edited KGN- lines
From: Balsalobre & Drouin, Nat Rev Molec Cell Biol, 2022.
Fig. 1 Priming of the enhancer region through binding of pioneer transcription factor to the closed chromatin
Objectives • To develop novel cell culture model systems and • To determine whether oncogenic Foxl2- C134W has pioneering activity in aGCTs. Methods • We used CRISPR/Cas9 editing to generate isogenic aGCT cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO) (Fig. 2). • ATAC-Seq and endogenous Foxl2 ChIP- Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. • ENCODE pipelines and data standards were used for analysis and the irreproducible discovery rate was used to identify high-reliability ATAC-seq and ChIP- seqpeaks. • De novo motifs were identified with the STREME algorithm.
Fig. 4A & B Chromatin accessibility in Foxl2 ChIP-Seq peaks by genotypes
1. Pilsworth, J.A., et al., Adult-type granulosa cell tumor of the ovary: a FOXL2-centric disease. J Pathol Clin Res, 2021. 7 (3): p. 243-252. 2. Shah, S.P., et al., Mutation of FOXL2 in granulosa-cell tumors of the ovary. N Engl J Med, 2009. 360 (26): p. 2719-29. 3. Carles, A., et al., The Pathognomonic FOXL2 C134W Mutation Alters DNA-Binding Specificity. Cancer Res, 2020. 80 (17): p. 3480-3491. 4. Taube, J.H., et al., Foxa1 functions as a pioneer transcription factor at transposable elements to activate Afp during differentiation of embryonic stem cells. J Biol Chem, 2010. 285 (21): p. 16135-44.
Fig. 2 Allele-specific CRISPR-Cas9 editing of FOXL2 in KGN cell linePage 1
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